Full Text:   <2526>

Summary:  <1641>

CLC number: S482.3+3

On-line Access: 2016-02-01

Received: 2015-01-08

Revision Accepted: 2015-07-06

Crosschecked: 2016-01-06

Cited: 0

Clicked: 4042

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Hong-cui Liu

http://orcid.org/0000-0002-3531-6246

Shao-nan Li

http://orcid.org/0000-0001-8158-7891

-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.2 P.110-126

http://doi.org/10.1631/jzus.B1500008


Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna


Author(s):  Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li

Affiliation(s):  Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China; more

Corresponding email(s):   snli@zju.edu.cn

Key Words:  Daphnia magna, Cholinesterase (ChE), Polymerase chain reaction (PCR), Recombinant protein ChE, Enzyme-linked immunosorbent assay (ELISA), Triazophos


Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna[J]. Journal of Zhejiang University Science B, 2016, 17(2): 110-126.

@article{title="Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna",
author="Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li",
journal="Journal of Zhejiang University Science B",
volume="17",
number="2",
pages="110-126",
year="2016",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1500008"
}

%0 Journal Article
%T Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
%A Hong-cui Liu
%A Bing-qiang Yuan
%A Shao-nan Li
%J Journal of Zhejiang University SCIENCE B
%V 17
%N 2
%P 110-126
%@ 1673-1581
%D 2016
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1500008

TY - JOUR
T1 - Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
A1 - Hong-cui Liu
A1 - Bing-qiang Yuan
A1 - Shao-nan Li
J0 - Journal of Zhejiang University Science B
VL - 17
IS - 2
SP - 110
EP - 126
%@ 1673-1581
Y1 - 2016
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1500008


Abstract: 
To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

大型溞胆碱酯酶蛋白的原核表达及其多克隆抗体的制备和适用性研究

目的:原核表达并纯化大型溞胆碱酯酶(ChE),制备鼠抗大型溞ChE多克隆抗体,并对抗体的适用性进行研究。
创新点:首次通过原核表达获得了大型溞重组ChE蛋白,通过免疫小鼠获得了高效价、高特异性的多克隆抗体,通过样品检测证明了抗体的适用性。
方法:利用聚合酶链式反应(PCR)技术获得大型溞ChE基因编码序列,并将其亚克隆至原核表达载体pET-29a(+),构建重组表达质粒pET-ChE,用异丙基硫代半乳糖苷(IPTG)诱导重组蛋白的表达;对表达产物进行聚丙烯酰胺凝胶电泳(SDS-PAGE)和酶活性检测,并对蛋白进行纯化(图4);使用纯化蛋白免疫BALB/c小鼠,制备多克隆抗体(图5),用酶联免疫吸附分析法(ELISA)对处于三唑磷和低温胁迫下以及经反复冻融处理的大型溞体内的ChE含量变化进行检测(图7、表6和7),以评价抗体的可适用性。
结论:获得了高纯度的ChE重组蛋白;免疫小鼠后,获得了特异性强、高效价的抗体;通过对处于三唑磷和低温胁迫下以及经过反复冻融处理的大型溞体内的ChE含量的检测,证明了抗体的适用性。

关键词:大型溞;胆碱酯酶;聚合酶链式反应(PCR);重组胆碱酯酶蛋白;酶联免疫吸附分析法(ELISA)

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]Abdel-Halim, K.Y., Salama, A.K., El-khateeb, E.N., et al., 2006. Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses. Chemosphere, 63(9):1491-1498.

[2]Anthony, N., Rocheleau, T., Mocelin, G., et al., 1995. Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti. FEBS Lett., 368(3):461-465.

[3]Barata, C., Baird, D.J., Soares, A.M.V.M., et al., 2001. Biochemical factors contributing to response variation among resistant and sensitive clones of Daphnia magna Straus exposed to ethyl parathion. Ecotoxicol. Environ. Saf., 49(2):155-163.

[4]Baslow, M.H., Nigrelli, R.F., 1964. The effects of thermal acclimation on brain cholinesterase of the killifish, Fundulus heteroclitus. Zoologica, 49:41-51.

[5]Botté, E.S., Smith-Keune, C., Jerry, D.R., 2013. Temperature: a prolonged confounding factor on cholinesterase activity in the tropical reef fish Acanthochromis polyacanthus. Aquat. Toxicol., 140-141:337-339.

[6]Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein dye binding. Anal. Siochem., 72(1-2):248-254.

[7]Cajaraville, M.P., Bebianno, M.J., Blasco, J., et al., 2000. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci. Total Environ., 247(2-3):295-311.

[8]Carvalho, F.D., Machado, I., Martinez, M.S., et al., 2003. Use of atropine-treated Daphnia magna survival for detection of environmental contamination by acetylcholinesterase inhibitors. Ecotoxicol. Environ. Saf., 54(1):43-46.

[9]Chen, L., Li, B., Pu, G.Q., 2010. Cloning and sequence analysis of cDNA fragment of acetylcholinesterase gene in Spodoptera litura. Sci. Sericult., 36(1):0138-0142.

[10]Coelho, S., Oliveira, R., Pereira, S., et al., 2011. Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels. Aquat. Toxicol., 103(3-4):191-198.

[11]Damásio, J., Guilhermino, L., Soares, A.M.V.M., et al., 2007. Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion. Chemosphere, 70(1):74-82.

[12]den Besten, P.J., Valk, S., van Weerlee, E., et al., 2001. Bioaccumulation and biomarkers in the sea star Asterias rubens (Echinodermata: Asteroidea): a North Sea field study. Mar. Environ. Res., 51(4):365-387.

[13]Denoyelle, R., Rault, M., Mazzia, C., et al., 2007. Cholinesterase activity as a biomarker of pesticide exposure in Allolobophora chlorotica earthworms living in apple orchards under different management strategies. Environ. Toxicol. Chem., 26(12):2644-2649.

[14]Diamantino, T.C., Almeida, E., Soares, A.M.V.M., et al., 2003. Characterization of cholinesterases from Daphnia magna Straus and their inhibition by zinc. Bull. Environ. Contam. Toxicol., 71(2):219-225.

[15]Duquesne, S., 2006. Effects of an organophosphate on Daphnia magna, at subspeciesal and speciesal levels: implications for population dynamics. Ecotoxicol. Environ. Saf., 65(2):145-150.

[16]Duquesne, S., Küster, E., 2010. Biochemical, metabolic, and behavioural responses and recovery of Daphnia magna after exposure to an organophosphate. Ecotoxicol. Environ. Saf., 73(3):353-359.

[17]Elendt, B.P., Bias, W.R., 1990. Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing: effects of the optimization of culture conditions on life history parameters of Daphnia magna. Water Res., 24(9):1157-1167.

[18]Gälli, R., Rich, H.W., Scholtz, R., 1994. Toxicity of organophosphate insecticides and their metabolites to the water flea Daphnia magna, the Microtox test and an acetylcholinesterase inhibition test. Aquat. Toxicol., 30(3):259-269.

[19]Garabrant, D.H., Aylward, L.L., Berent, S., et al., 2009. Cholinesterase inhibition in chlorpyrifos workers: characterization of biomarkers of exposure and response in relation to urinary TCPy. J. Expo. Sci. Environ. Epidemiol., 19(7):634-642.

[20]Guilhermino, L., Lopes, M.C., Carvalho, A.P., et al., 1996. Inhibition of acetylcholinesterase activity as effect criterion in acute tests with juvenile Daphnia magna. Chemosphere, 32(4):727-738.

[21]Hackenberger, B.K., Jarić-Perkušić, D., Stepić, S., 2008. Effect of temephos on cholinesterase activity in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae). Ecotoxicol. Environ. Saf., 71(2):583-589.

[22]Hall, L.M.C., Malcolm, C.A., 1991. The acetylcholinesterase gene of Anopheles stephensi. Cell. Mol. Neurobiol., 11(1):131-141.

[23]Hill, E.F., 1989. Sex and storage affect cholinesterase activity in blood plasma of Japanese quail. J. Wildl. Dis., 25(4):580-585.

[24]Hogan, J.W., 1970. Water temperature as a source of variation in specific activity of brain acetylcholinesterase of bluegills. Bull. Environ. Contam. Toxicol., 5(4):347-353.

[25]Jemec, A., Drobne, D., Tisler, T., et al., 2007. The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test. Comp. Biochem. Physiol. C: Toxicol. Pharmacol., 144(4):303-309.

[26]Jiang, H.B., Liu, S.W., Zhao, P.C., et al., 2009. Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae. Insect Biochem. Mol. Biol., 39(9):646-653.

[27]Kaufer, D., Friedman, A., Seidman, S., et al., 1999. Anticholinesterases induce multigenic transcriptional feedback response suppressing cholinergic neurotransmission. Chem. Biol. Interact., 119-120:349-360.

[28]Key, P.B., Fulton, M.H., 2002. Characterization of cholinesterase activity in tissues of the grass shrimp (Palaemonetes pugio). Pestic. Biochem. Physiol., 72(3):186-192.

[29]Khattab, A.D., Ali, L.S., 2007. Immunoassays for avian butyrylcholinesterase: implications for ecotoxicological testing and clinical biomonitoring. Environ. Toxicol. Pharmacol., 24(3):275-285.

[30]Khattab, A.D., Walker, C.H., Johnston, G., et al., 1994. An ELISA assay for avian serum butyrylcholinesterase: a biomarker for organophosphates. Environ. Toxicol. Chem., 13(10):1661-1667.

[31]Kondo, M., Hada, T., Fukui, K., et al., 1995. Enzyme-linked immunosorbent assay (ELISA) for Aleuria aurantia lectin-reactive serum cholinesterase to differentiate liver cirrhosis and chronic hepatitis. Clin. Chim. Acta, 243(1):1-9.

[32]Li, C.X., Dong, Y.D., Liu, M.D., et al., 2007. Alternative splicing of ace1 gene in Culex pipiens pallens and its effect to enzyme activity. Acta Parasitol. Med. Entomol. Sin., 14(3):153-157 (in Chinese).

[33]Li, F., Han, Z.J., 2002. Cloning and sequencing of two acetylcholinesterase cDNA fragments from cotton aphid, Aphis gossypii Glover. Zool. Res., 23(5):444-448 (in Chinese).

[34]Li, S.N., Tan, Y.J., 2011. Hormetic response of cholinesterase from Daphnia magna in chronic exposure to triazophos and chlorpyrifos. J. Environ. Sci., 23(5):852-859.

[35]Li, S.N., Xie, X.C., Tan, Y.J., et al., 2005. Induction of triazophos to brain acetylcholinesterase from topmouth gudgeon, Pseudorasbora parva. Chin. J. Pest. Sci., 7(1):59-62 (in Chinese).

[36]Lin, T., Li, L.N., Chang, R.L., et al., 2007. Cloning and sequence analysis of acetylcholinease gene of Pseudophacopteron canarium. J. Jilin Agric. Univ., 29(4):368-370 (in Chinese).

[37]Liu, H.C., Yuan, B.Q., Li, S.N., 2012a. Altered quantities and in vivo activities of cholinesterase from Daphnia magna in sub-lethal exposure to organophosphorus insecticides. Ecotoxicol. Environ. Saf., 80:118-125.

[38]Liu, H.C., Yang, Y.X., Li, S.N., 2012b. Quantitative analysis of cholinesterase from Daphnia magna by indirect and non-competitive enzyme-linked immunosorbent assay. J. Zhejiang Univ. (Agric. Life Sci.), 38(3):347-354 (in Chinese).

[39]Monserrat, J.M., Bianchini, A., 1998. Some kinetic and toxicological characteristics of thoracic ganglia cholinesterase of Chasmagnathus granulate (Decapoda, Grapsidae). Comp. Biochem. Physiol. C: Toxicol. Pharmacol., 120(2):193-199.

[40]Ni, X.Y., Tomita, T., Kasai, S., et al., 2003. cDNA and deduced protein sequence of acetylcholinesterase from the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Appl. Entomol. Zool., 38(1):49-56.

[41]Phillips, T.A., Summerfelt, R.C., Atchison, G.J., 2002. Environmental, biological, and methodological factors affecting cholinesterase activity in walleye (Stizostedion vitreum). Arch. Environ. Contam. Toxicol., 43(1):75-80.

[42]Printes, L.B., Fellowes, M.D.E., Callaghan, A., 2008. Clonal variation in acetylcholinesterase biomarkers and life history traits following OP exposure in Daphnia magna. Ecotoxicol. Environ. Saf., 71(2):519-526.

[43]Printes, L.B., Fernandes, M.N., Espíndola, E.L.G., 2011. Laboratory measurements of biomarkers and individual performances in Chironomus xanthus to evaluate pesticide contamination of sediments in a river of southeastern Brazil. Ecotoxicol. Environ. Saf., 74(3):424-430.

[44]Rattner, B.A., 1982. Diagnosis of anticholinesterase poinsoning in birds: effects of environmental temperature and underfeeding on cholinesterase activity. Environ. Toxicol. Chem., 1(4):329-335.

[45]Sáenz, L.A., Seibert, E.L., Zanette, J., et al., 2010. Biochemical biomarkers and metals in Perna perna mussels from mariculture zones of Santa Catarina, Brazil. Ecotoxicol. Environ. Saf., 73(5):796-804.

[46]Sanchez-Hernandez, J.C., Fossi, M.C., Leonzio, C., et al., 1998. Use of biochemical biomarkers as a screening tool to focus the chemical monitoring of organic pollutants in the Biobio river basin (Chile). Chemosphere, 37(4):699-710.

[47]Scaps, P., Borot, O., 2000. Acetylcholinesterase activity of the polychaete Nereis diversicolor: effects of temperature and salinity. Comp. Biochem. Physiol. C: Pharmacol. Toxicol. Endocrinol., 125(3):377-383.

[48]Stien, X., Percic, P., Gnassia-Barelli, M., et al., 1998. Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea. Environ. Pollut., 99(3):339-345.

[49]Sturm, A., Hansen, P.D., 1999. Altered cholinesterase and monooxygenase levels in Daphnia magna and Chironomus riparius exposed to environmental pollutants. Ecotoxicol. Environ. Saf., 42(1):9-15.

[50]Sturm, A., Wogram, J., Hansen, P.D., et al., 1999. Potential use of cholinesterase in monitoring low levels of organophosphates in small streams: natural variability in three-spined stickleback (Gasterosteus aculeatus) and relation to pollution. Environ. Toxicol. Chem., 18(2):194-200.

[51]van Oosterom, J., King, S.C., Negri, A., et al., 2010. Investigation of the mud crab (Scylla serrata) as a potential bio-monitoring species for tropical coastal marine environments of Australia. Mar. Pollut. Bull., 60(2):283-290.

[52]Vesela, S., Kuca, K., Jun, D., 2006. Toxicity of the nerve agent tabun to Daphnia magna, a new experimental species in military toxicology. Chem. Ecol., 22(2):175-180.

[53]Villatte, F., Bachmann, T.T., 2002. How many genes encode cholinesterase in arthropods Pestic. Biochem. Physiol., 73(2):122-129.

[54]Xuereb, B., Noury, P., Felten, V., et al., 2007. Cholinesterase activity in Gammarus pulex (Crustacea Amphipoda): characterization and effects of chlorpyrifos. Toxicology, 236(3):178-189.

[55]Xuereb, B., Chaumot, A., Mons, R., et al., 2009. Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): intrinsic variability, reference levels, and a reliable tool for field surveys. Aquat. Toxicol., 93(4):225-233.

[56]Yang, L.G., Hu, S.C., Wei, P.H., et al., 1998. Enzyme Immunoassay. Nanjing University Press, Nanjing, p.279-281 (in Chinese).

[57]Yang, Y.X., 2010. Immunoassays for Daphnia magna cholinesterases: a biomarker for organophosphates. Master’s Thesis, Zhejiang University, Hangzhou, p.1-95 (in Chinese).

[58]Yang, Y.X., Niu, L.Z., Li, S.N., 2013. Purification and studies on characteristics of cholinesterases from Daphnia magna. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 14(4):325-335.

[59]Zhang, T., 2008. Expression of Drosophila melanogaster acetylcholinesterase (AchE) gene in Pichia pastoris. Master’s Thesis, Chongqing University, Chongqing, p.1-52 (in Chinese).

[60]Zhou, M.J., Zhang, C.L., Richard, P., et al., 2000. Choline oxidase: a useful tool for high-throughput assays of acetylcholinesterase, phospholipase D, phosphatidylcholine-specific phospholipase C, and sphingomyelinase. Proc. SPIE, 3926:166-171.

Open peer comments: Debate/Discuss/Question/Opinion

<1>

Please provide your name, email address and a comment





Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE